Compounds and methods for diagnosing and treating amyloid-related conditions

ABSTRACT

The invention provides methods for diagnosing and treating amyloid-related conditions and compounds useful for the same. The invention provides for detecting, imaging, monitoring, diagnosing, and treating conditions characterized by the binding or aggregation of amyloid fibrils. More particularly, the invention relates to using quinolinehydrazone compounds for diagnosing and treating amyloidotic conditions and also as an antioxidant.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention provides methods for diagnosing and treatingamyloid-related conditions and compounds useful for the same. Theinvention allows for detecting, imaging, monitoring, diagnosing, andtreating conditions characterized by the binding or aggregation ofamyloid fibrils. More particularly, the invention relates to the use ofquinolinehydrazone compounds for diagnosing and treating amyloidoticconditions.

[0003] The invention also encompasses a method for usingquinolinehydrazone compounds as an antioxidant.

[0004] 2. Description of the Related Technology

[0005] Disease-causing agents once were believed to be limited topathogens containing nucleic acids, for example viruses and bacteria.More recently, however, considerable evidence suggests that irregularpeptides or proteinaceous infective agents can induce or transmitinfectious disease.

[0006] Currently, it is widely recognized that some proteins can containirregular protein sequences which cause living tissue or organs toassemble into insoluble aggregates of partially unfolded proteins, knownas amyloid fibrils. All types of amyloid are structurally related bycontaining Aβ peptides. The Aβ peptides aggregate to form fibrils, whichtypically have a β-sheet secondary structure. The fibril deposits, orplaque, are believed to be at the root of the pathology for a number ofneurodegenerative diseases, or amyloidosis.

[0007] In general, the term “amyloidosis” refers to diseasescharacterized by the tendancy of particular proteins to aggregate andprecipitate as insoluble fibrils. The fibrils collect in theextracellular space of the surrounding organs or tissues causingstructural and functional damage. Common attributes of amyloidosisinclude, for example, cell toxicity and cell degeneration. For instance,the clinical course of the amyloidotic condition, Alzheimer's disease,is neurodegeneration. Neurodegeneration can be identified, for example,by the progressive loss of mental capacity, loss of motor control, anddementia. Portmortem deposits of amyloid plaque have been identified inpatients suffering from Alzheimer's disease, Down syndrome, Type 2diabetes mellitus, and other amyloid-related conditions.

[0008] Infectious protein particles, or prions, also contain irregularprotein sequences. These particles typically can be characterized by asingle irregular sequence in the protein peptide. The expression of theirregular sequence favors a protein conformation that, like amyloidprotein, tends to aggregate. The aggregation of the protein into aplaque has been identified in many patients inflicted with priondisease, such as, for example, Crutzfeld-Jacob disease (CJD),Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia(FFI), kuru, Alper's syndrome, scrapie, transmissible minkencepthalopathy (TME), chronic wasting disease (CWD), and bovinespongiform encephalopathy (BSE). In some cases, amyloid plaque has beendetected in patients having prion-initiated disease, for examplepatients diagnosed with CJD. However, prion plaque generally has notbeen definitively linked to cell impairment. Instead, considerableevidence suggests that the prions propagate by changing the conformationof naturally-existing, non-infectious counterpart proteins into proteinshaving a harmful secondary structure.

[0009] Few methods are known for identifying, treating, or inhibitingthe aggregation of amyloid proteins or prions. The target proteins orplaque often exhibit similar properties as healthy, unaffected tissue.As a result, it is difficult to develop an imaging agent selective foronly harmful plaque or protein.

[0010] Moreover, the harmful plaque often resides in the brain, forexample in the case of Alzheimer's disease. To provide a method that isuseful for associating with brain plaque, the compound will have usefulproperties both for binding to amyloid or prion plaque and for crossingthe blood-brain barrier. Compounds useful for staining or imaging theplaque in vitro often can not cross the highly discriminant blood-brainbarrier to provide a useful in vivo diagnostic or therapeutic tool.Consequently, successful compounds and methods for detecting plaque invitro can fail as a tool for in vivo imaging.

[0011] One compound useful for selectively identifying plaque in vitrois the commercially available diazo dye, Congo red, having thescientific name3,3′-[[1,1′-biphenyl]-4,4′-diylbis(azo)]bis[4-amino-1-naphthalenesulfonicacid] disodium salt. Congo red has demonstrated binding to amyloid-likeproteins with a beta-pleated sheet conformation. See, W. E. Klunk, etal., Quantitative Evaluation of Congo Red Binding to Amyloid-likeProteins with a Beta-Pleated Sheet Conformation, J. Histochem.Cytochem., 37:1273-1281 (1989). However, Congo red lacks the necessaryproperties to cross the blood-brain barrier as shown by P. D. Griffiths,et al., Receptor Changes in the Neocortex of Postmortem Tissue inParkinson's Disease and Alzheimer's Disease, Dementia, 3:239-246 (1992).As such, the compound provides useful properties for postmortem stainingof brain plaque in vitro, but is unsuitable for in vivo use.

[0012] Chrysamine G (CG) compounds are a class of compounds derived fromCongo red. The predominant structural difference is replacement ofsulphonic acid groups in Congo red with carboxylic acid groups. See, forexample, International Publication Nos. WO 98/47969, published Oct. 29,1998; WO 96/34853, published Nov. 7, 1996; and WO 99/24394, publishedMay 20, 1999. In addition, organometallic ligands of Congo red andChrysamine G compounds have been investigated for use as a diagnostictool. See, for example, International Publication No. WO 97/41856,published Nov. 13, 1997.

[0013] The replacement of sulphonic acid groups with carboxylic acidgroups potentially would effect better blood-brain barrier entry of thecompound. However, in vivo biodistribution of Chrysamine G compounds hasshown that technetium-99m-labeled conjugates of 2-(acetamido)-CG withbis-S-trityl protected monoamide-monoaminedithiol were rapidly clearedfrom the blood, causing low uptake of the conjugate in the brain. See,for example, N. A. Dezutter, European Journal of Nuclear Medicine, Vol.26, No. 11, pp. 1392-1399 (1999); W. E. Klunk, et al., Life Sciences,Vol. 63, No. 20, pp. 1807-1814 (1998); and W. E. Klunk, et al.,Neurobiology of Aging, Vol. 16, No. 4, pp. 541-548 (1995).

[0014] Derivatives of 9-acridinone also have been reported as inhibitingamyloid aggregation. International Publication No. WO 97/16191,published May 9, 1997, describes that 9-acridinone compounds inhibitamyloid aggregation in vitro. In vivo diagnostic or therapeutic activityof the compounds is not described.

[0015] Commonly-assigned U.S. Pat. Nos. 5,731,313, and 5,998,615, issuedMar. 24, 1998, and Dec. 7, 1999, respectively, identifyfluoroanthracyclinone compounds as useful anti-infective agents and alsoas a diagnostic agent for imaging amyloid plaque. The in vitro bindingof Aβ25-35 peptides is described in International Publication No. WO97/49433, published Dec. 31, 1997.

[0016] More recently, naphthylazo derivatives have been prepared forinhibiting amyloid aggregation. The in vitro binding activity ofnaphthylazo compounds is described in International Publication No.97/16194, published May 9, 1997. However, no in vivo data is describedin the publication.

[0017] Radiolabeled hydrazine and ethylene derivatives ofbenzenethiazole compounds also have been reported for binding insulinamyloid. See, International Publication WO 97/26919, published Jul. 31,1997. These compounds show inhibitory activity in vitro, however, to thebest of our knowledge, no in vivo activity has been shown.

[0018] Of the methods known, the only conclusive test is staining forplaque postmortem. Some compounds have been investigated for in vivoadministration and imaging, but it remains beneficial to provide acompound for demonstrating in vitro imaging of compounds.

[0019] Accordingly, it remains beneficial to provide a compound fordemonstrating in vitro and in vivo imaging of harmful, proteinaceousplaque or infectious agents, more particularly amyloid plaque, prions,or prion plaque. There remains a need to identify compounds and methodsfor diagnosing and treating conditions related to the aggregation ofamyloid or other proteinaceous fibrils. Described here are compoundsthat provide in vitro imaging, diagnostic opportunities, antioxidantproperties and even treatment opportunities for patients suffering fromamyloidotic conditions.

SUMMARY OF THE INVENTION

[0020] The invention provides diagnostic and therapeutic methods relatedto detecting, imaging, monitoring, diagnosing, and treating conditionsrelated to aggregation of amyloid or other harmful proteinaciousdeposits and compounds for accomplishing the methods. Compoundsdemonstrating beneficial properties for the methods of the inventiongenerally are quinolinehydrazone compounds having the formula:

[0021] or a pharmaceutically acceptable salt, ester, solvate or prodrugthereof, wherein:

[0022] R¹, R², R³, R⁴, and R⁵ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; and

[0023] R⁶ is a benzopyridinyl group optionally substituted with one tothree substituents selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, triflouromethyl, trifluoromethylether, halo,and a group of the formula —OR⁷, wherein R⁷ is alkyl or aryl;

[0024] wherein said alkyl groups at each occurrence are optionallysubstituted with alkoxy, aryl, or halo; and said aryl groups at eachoccurrence are optionally substituted with alkyl, alkoxy, or halo. Oneor more atoms in the compound can be optionally replaced with aradiolabeled atom, or radioisotope (radioactive isotope). Theradioisotope can be selected from the group consisting of ³H, ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F, ¹⁵O, and ¹¹C. Pharmaceuticallyacceptable salts, esters, solvates, or prodrugs of compounds having theformula (I) can also be suitable for methods of the invention.

[0025] In one aspect, therefore, the invention relates to a method forchemically tagging or inhibiting the aggregation of amyloid fibrilscomprising administering an effective amount of a compound of formula(I) and allowing the compound to associate with the amyloid fibrils. Inthis manner, the compound prevents amyloid fibrils from binding to eachother and, accordingly, arrests the formation of harmful amyloid plaque.

[0026] In another aspect, the invention relates to a method fordetecting aggregation of amyloid fibrils. The method comprises (a)administering a compound of formula (I) containing at least oneradioactive isotope; (b) allowing the compound to associate with theamyloid fibrils to provide a labeled deposit; and (c) detecting thelabeled deposit. The detection method also can be used for monitoringthe aggregation of amyloid fibrils in an individual. If necessary orappropriate, data obtained from detecting the labeled amyloid deposit,such as the location and the amount of amyloid deposit, can be recordedto allow one with skill in the art to assess the status and/or progressof the amyloidotic condition.

[0027] The method also can be used for identifying or diagnosing acondition characterized by aggregation of amyloid fibrils, furthercomprising evaluating or assessing the amount and placement of theradioactive isotope to determine the medical condition of theindividual, or patient. To achieve the full advantage of the invention,the data can be evaluated or assessed in light of data obtained from anormal, i.e. free of amyloid-plaque, individual. In this aspect, themethod comprises identifying or using a biomarker for conditionscharacterized by the aggregation of amyloid fibrils. In addition, themethod can also be used in a patient undergoing treatment for acondition characterized by the aggregation of amyloid fibrils toevaluate the progress of the course of treatment.

[0028] In another aspect, the invention relates to a method for treatinga condition characterized by aggregation of amyloid fibrils comprisingadministering a compound of formula (I), optionally in apharmaceutically acceptable carrier, and allowing the compound toassociate with the amyloid plaque. The treatment can be carried out byallowing the compound to directly bind to or inhibit the aggregation ofthe amyloid plaque. The steps of administering the compound and allowingit to associate can be repeated as necessary or desired to effectuatetreatment of the amyloid-related condition.

[0029] Alternatively, the compound can deliver therapeutic agents to theamyloid fibrils. The method for delivering a treatment for a conditioncharacterized by aggregation of amyloid fibrils, therefore comprises (a)providing a compound of formula (I) in combination with a therapeuticagent; (b) administering the combination to an individual havingamyloidosis; and (c) optionally repeating steps (a) and (b), asnecessary, to improve or rehabilitate the condition of the individual.

[0030] Yet another aspect of the invention relates to a method forstaining amyloid fibrils comprising (a) applying a compound of formula(I) to a sample containing amyloid fibrils to form a labeled deposit and(b) detecting the labeled deposit.

[0031] In addition, the invention can relate to a method for detectingamyloid deposits in biopsy or postmortem human or animal tissuecomprising the steps of (a) incubating formalin-fixed tissue with asolution of a compound of formula (I) to form a labeled deposit and (b)detecting the labeled deposit.

[0032] The compounds also can be contacted with bodily fluids extractedfrom a mammal to detect aggregated prion proteins. In this aspect, abodily fluid is extracted from the mammal and contacted with the bodilyfluid of a compound of formula (I).

[0033] In yet another aspect, a quinolinehydrazone compound can be usedas an antioxidant by administering the compound, optionally in apharmaceutically acceptable carrier. The quinolinehydrazone compound cancomprise a compound of formula (I).

[0034] Yet another aspect of the invention relates to a complexcomprising a compound of formula (I) in association with or bound to anamyloid fibril. The complex also can comprise a compound of formula (I)in association with or bound to a proteinaceous infectious particle, orprion.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Quinolinehydrazone compounds possess beneficial properties forproviding novel diagnostic and therapeutic methods for detecting,imaging, monitoring, diagnosing, and treating conditions related toaggregation of amyloid and other harmful proteinacious deposits.Preferred quinolinehydrazone compounds comprise:

[0036] or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein R¹, R², R³, R⁴, R^(5,) and R⁶ are as previouslydefined, and wherein one or more atoms of the compound optionally can besubstituted with a radiolabeled atom.

[0037] The term “alkyl” as used herein refers to a straight or branchedhydrocarbon group, preferably containing one to six carbon atoms.Examples of useful alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (1-methylpropyl), t-butyl(1,1-dimethylethyl), n-pentyl, t-pentyl (1,1-dimethylpropyl), n-hexyl,and the like. Alkyl groups for the invention also can include groupswherein the hydrocarbon contains one or more substituents such as, forexample, cycloalkyl, alkoxy, aryl, or halo as defined hereinbelow. Thesubstituents can bond with the same carbon or different carbons.Typically, the hydrocarbon contains one, two or three substituents, ifany.

[0038] The term “alkoxy” as used herein refers to a straight or branchedhydrocarbon group as defined above attached to the parent moleculethrough an oxygen heteroatom, typically by a carbon to oxygen bond. Thehydrocarbon of the alkoxy group preferably contains from 1 to 6 carbonatoms. Typical alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, sec-butoxy (1-methylpropoxy), t-butoxy (1,1-dimethylethoxy),n-pentoxy, t-pentoxy (1,1-dimethylpropoxy), and the like.

[0039] The term “cycloalkyl” as used herein refers to non-aromaticcyclic hydrocarbon group, preferably containing from three to six carbonatoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. Cycloalkyl groups may be substituted withalkyl and alkoxy groups, as defined above, as well as halo substituents,for example, bromo, chloro, iodo, and fluoro.

[0040] The term “aryl” as used herein refers to an aromatic cyclichydrocarbon, such as phenyl and naphthyl. The aryl group optionally canbe substituted with alkyl, alkoxy or a halo group, for example, bromo,chloro, iodo, and fluoro. Examples of aryl groups include, but are notlimited to, phenyl, bromophenyl, chlorophenyl, iodophenyl, fluorophenyl,bromonaphthyl, and the like.

[0041] The term “triflouromethyl” as used herein refers to a methylgroup (—CH₃) wherein each hydrogen atom is substituted with a fluorineatom. Similarly, “trifluoromethylether” is a trifluoromethyl group, asdefined above, linked to the parent molecule through an oxygenheteroatom, typically by a carbon-oxygen bond. The trifluoromethyl andtrifluoromethylether groups can be represented by the formulae —CF₃ and—OCF₃, respectively.

[0042] The term “halo” as used herein refers to a monovalent substituentderived from a halogen. Typical halogens are, for example, bromine,chlorine, iodine, fluorine, and the like.

[0043] The term “benzopyridinyl” as used herein refers to a groupderived from a compound comprising a fused benzene and pyridine ring,generally called a benzopyridine. Examples of benzopyridine compoundsare quinoline and isoquinoline. The corresponding groups derived fromquinoline and isoquinoline are quinolyl and isoquinolyl, respectively.The benzopyridinyl groups can contain one or more substituents,typically selected from alkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl.

[0044] The term “radiolabeled atom”, “radioisotope”, or “radioactiveisotope” as used herein refers to an atom, which can be incorporatedinto a compound, capable of emitting waves or particles of radioactiveenergy, typically light and/or heat. More particularly, the atoms decayfrom a radioactive atom to a naturally occurring atom containing thesame number of protons by emitting electrons. Examples of radiolabeledatoms or radioisotopes are ³H, ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F,¹⁵O, and ¹¹C.

[0045] It has now been discovered that quinolinehydrazone compounds,particularly quinoline- or isoquinoline-substituted quinolinehydrazonecompounds, exhibit antioxidant properties and can bind to amyloidfibrils, inhibiting the aggregation thereof. The compounds demonstratein vitro activity for inhibiting seeded aggregation of amyloid fibrilsas well as demonstrate in vitro antioxidant properties in oxidativeneuronal models. Moreover, the compounds can cross the blood-brainbarrier in vivo to provide a beneficial diagnostic or therapeutic tool.

[0046] Certain quinolinehydrazone compounds previously have described asantimicrobial, antiparasitic, anti-mycoplasmal, and anti-HIV agents.See; for example, Atti Accad. Fisiocritici Siena, Serie XIV, Vol. 8,82-93 (1976); Arzneim.-Forson. (Drug Res.) 23, Nr. 6, 830-839 (1973); IlFarrmaco, 47(6), 945-962 (1992); and U.S. Pat. No. 3,646,019. However,quinolinehydrazone compounds reported in the literature typically aresubstituted with monocyclic, if any, heteroaryl groups. There is noknown description of using quinolinehydrazine compounds for binding toamyloid fibrils or inhibiting the aggregation of such fibrils either invitro or in vivo. In addition, quinoline- and isoquinoline-substitutedquinolinehydrazone compounds have not been described as a compound perse, either for demonstrating therapeutic activities or as a diagnostictool.

[0047] The compounds can be prepared by any suitable process. Thecompounds can be prepared from 2-hydrazinochloroquinoline startingmaterials or, in the alternative, prepared by reacting the corresponding2-chloroquinoline with hydrazine hydrate. The 2-hydrazinoquinoline groupis reacted with the carboxaldehyde of the desired benzopyridylsubstituent, for example 4-quinolinecarboxaldehyde. The reaction ispreferably carried out in an organic solvent, typically alcoholicsolvents, for example, methanol, ethanol, isopropanol, and the like. Themost preferred organic solvent is ethanol. To facilitate preparation,the compounds can be reacted in the presence of heat, typically fromabout 50-70° C. Additional discussion of the methods, reagents andconditions for preparing the quinolinehydrazone derivatives, in general,are described in Atti Accad. Fisiocritici Siena, Serie XIV, Vol. 8,82-93 (1976); Arzneim.-Forson. (Drug Res.) 23, Nr. 6, 830-839 (1973); IlFarmaco, 47(6) , 945-962 (1992).

[0048] Radiolabeled compounds can be prepared by reacting a compound offormula (I) with a radioactive isotope or a suitable reagent containingthe isotope. A thorough discussion regarding methods for preparingradiolabeled compounds and derivatives is provided in Kirk-Othmer,Encyclopedia of Chemical Technology, 4^(th) ed., vol. 20, John Wiley &Sons, Inc., New York, N.Y. (1999), and more particularly in pp. 930-962.

[0049] A compound of formula (I) can bind to Aβ peptide sequences with adissociation constant (Kp) between 0.001 and 10.0 μM when measured bybinding to synthetic Aβ peptide or to amyloid fibrils in brain tissue.In addition, the compound is capable of traversing capillary endothelialcells with continuous junctions and no detectable transendothelialpathways to reach the brain, commonly called the blood-brain barrier,which is beneficial for a diagnostic tool if used in the brain.Dose-dependent concentrations of the compound can be detected in thebrain and plasma of C57 female mice when administered in vivo, whichindicates transport across the blood-brain barrier.

[0050] Preferred compounds for the methods of the invention can berepresented by the general formula (I), or can comprise a salt, ester,solvate, or prodrug thereof, having at least one atom of the originalparent compound replaced with a radioactive isotope. The radioactiveisotopes emit wave frequencies, which are typically readily detectableby a variety of detectors or imagers. The scope of the inventioncontemplates detectors and imagers primarily functioning on thedetection of wave frequencies, for example photon emissions, positronemissions, gamma emissions, and the like. The detector and imagingapparatus suitable for use with the invention is not limited bycommercial availability of the units, however, many practical units fordetecting, imaging, possibly in combination with recording, the presenceof the radioactive isotopes in the body of the mammal are readilyavailable. More particularly, non-invasive units for detecting andimaging radioactive isotopes in vivo, generally in combination withelectronic, photographic, or textual methods of recording data, are alsocontemplated. The detector or imaging equipment detect wave frequenciesemitted from the labeled compounds that can be distinguished from othernon-labeled compounds, i.e. compounds not containing a radioactiveisotope. It is well within the purview of one skilled in the art todetermine the particular radioactive isotope for an intended detectionor imaging assay, considering the frequency range and sensitivity of thedetector, the chemical and physical properties of the imaging agent, andother factors relevant to the precision, accuracy, and validity of theassay.

[0051] The preferred substituents for R¹, R² , R³, R⁴, and R⁵ are eachindependently selected from hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl, n-hexyl,methoxy, ethoxy, isopropoxy, sec-butoxy, t-butoxy, phenyl, benzyl,trifluoromethyl, trifluoromethylether, or halo. The preferred group forthe substituent R⁶ in a compound of formula (I) is quinolyl orisoquinolyl. More preferred compounds for the methods of the inventionhave the formula:

[0052] wherein R⁸ and R⁹ are as previously defined for the substituentsR¹, R², R³, R⁴, and R⁵ ₁ or a pharmaceutically acceptable salt, ester,solvate, or prodrug thereof. The more preferred substituents for R⁸ andR⁹ are each independently selected from hydrogen, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl,n-hexyl, methoxy, ethoxy, isopropoxy, sec-butoxy, t-butoxy, phenyl,benzyl, trifluoromethyl, trifluoromethylether, or halo. Preferredradiolabeled atoms are ³H, and ¹²⁵I.

[0053] More particularly, the more preferred compounds for the inventioninclude, but are not limited to:

[0054] 4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;

[0055] 4-ethyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;

[0056] 4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;

[0057] 4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;

[0058] 4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;

[0059] 4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline; and

[0060] 4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.

[0061] The term “pharmaceutically acceptable salt, ester, solvate, orprodrug thereof” refers to derivatives prepared from a parent compound,in this case the quinolinehydrazone compound. The salts include, but arenot limited to, alkali metal salts, alkaline earth metal salts, ammoniumsalts, salts with an appropriate organic amine or amino acid, and acidaddition salts prepared with organic or inorganic acids such as, forexample, hydrochloric acid, sulfuric acid, carboxylic acid, and sulfonicacids. Esters more particularly refer to condensation products of theparent compound with an organic acid, for example, acetate, propionate,butyrate, valerate, benzoate, salicylate, succinate, and the like.Solvates refer to ionic or molecular complexes Comprising the parentcompound and one or more molecules of solute, typically an organicsolvent, and preferably water. Prodrugs are obtained in vivo when achemical compound is converted into a parent compound, for example aquinoline- or isoquinoline-substituted quinolinehydrazone, by naturalmetabolic processes of the body.

[0062] When administered to a mammal, the compounds have demonstrated anability to interact with amyloid fibrils, and particularly with amyloidfibrils in the brain. To achieve the full advantage of the invention,the compound is allowed to interact with the amyloid fibrils, generallyby allowing a sufficient time for bonding, associating, conjugating,complexing, or other reaction to take place. In the presence of amyloidfibrils, the labeled compounds interact with the amyloid fibrils to forma labeled amyloid fibril complex. In particular, the radiolabeledcompound can be used to bind to brain amyloid fibrils including, forexample, protofibrils, type-1 fibrils, type-2 fibrils, neuritic plaque,diffuse amyloid, and combinations thereof. In some cases, the compoundmay interact with only one type of fibril, either by nature or bydesign. In a preferred case, the compound specifically interacts withβ-amyloid protofibrils and, more specifically, β-amyloid protofibrils inthe brain.

[0063] For the convenience and ease of administration, the compoundcommonly is prepared into a pharmaceutical composition. In a typicalpharmaceutical composition, a quinolinehydrazone compound or aderivative thereof, including a radiolabeled compound preparedtherefrom, as described above, is combined with a pharmaceuticallyacceptable carrier. The carrier can comprise one or more solubilizingagents, excipients, additives, diluents, or other agents to improve thecharacteristics of the composition. In general, the compound isformulated with the carrier in any manner suitable for providing apharmaceutical dosage form, typically resulting in a sterile, non-toxicmixture or blend of the active agent with a carrier. The composition canbe administered orally, parenterally, subcutaneously, intravenously,intramuscularly, intracisternally, or by infusion techniques. Methodsfor preparing pharmaceutical compositions are readily available in theart and are further described, for example, in Remington: The Scienceand Practice of Pharmacy, Nineteenth Edition, vol. II, Mack PublishingCompany, Easton, Pa., 1995, in particular with respect to solutions,emulsions, suspensions, and extracts on pp. 1485-1533; intravenousadmixtures on pp. 1542-1562; oral dosage forms on pp. 1615-1649;sustained-release delivery systems on pp. 1660-1675; and aerosols on pp.1676-1692.

[0064] In particular, the compounds can be administered in a liquid,solid, or semi-solid oral dosage form. Examples of common dosage formssuitable for the invention are tablets, capsules, troches, lozenges,emulsions, solutions, tinctures, syrups, elixirs, and the like. Oraldosage forms can optionally contain one or more chemically non-reactiveexcipients, for example, sweetening agents, flavoring agents, coloringagents, preserving agents, and the like.

[0065] The active agent can be admixed with excipients, such as inertdiluents, granulating and disintegrating agents, binding agents, andlubricating agents to provide a suitable tablet. Examples of inertdiluents suitable for the tablet are, for example, calcium carbonate,sodium carbonate, lactose, calcium phosphate and sodium phosphate.Granulating and disintegrating agents suitable for the inventioninclude, but are not limited to, maize starch, alginic acid, and thelike. Binding agents, for example maize starch, gelatin or acacia, canalso be incorporated into the tablet. Lubricating agents, such as, forexample magnesium stearate, stearic acid or talc, are also suitable forthe invention.

[0066] The tablets can be optionally coated by a wide variety oftechniques to facilitate administration, improve taste, provide asustained action over a longer period, or delay disintegration oradsorption in the gastrointestinal tract. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. Techniques for coating tablets, in general, are furtherdescribed in Coating of Pharmaceutical Dosage Forms by Stuart C. Porter,reprinted in Remington: The Science and Practice of Pharmacy, NineteenthEdition, vol. II, Mack Publishing Company, Easton, Pa., 1995, inparticular in pp. 1650-1659.

[0067] Capsules are another dosage form suitable for oraladministration. The term “capsules” is intended to include hardcapsules, soft capsules such as gel capsules, microcapsules, and thelike. The active ingredient can be mixed with an inert solid diluent,for example, calcium carbonate, calcium phosphate or kaolin, or a softgelatin capsule wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.

[0068] Aqueous suspensions can contain the active materials in admixturewith one or more compatible excipients. Such excipients are suspendingagents, for example, sodium carboxymethylcellulose, methylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents such as naturally-occurring phosphatides, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for example,heptadecaethylene-oxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol anhydrides, for example polyoxyethylene sorbitan monooleate. Theaqueous suspensions may also contain one or more preservatives, forexample, ethyl or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, or one or more sweetening agents,such as sucrose or saccharin.

[0069] In addition to aqueous suspensions, the active agent can beformulated as an oily suspension. The oily formulation is prepared bysuspending the active ingredient in a vegetable oil, for example arachisoil, olive oil, sesame oil, coconut oil, or mineral oil, such as liquidparaffin. The oily suspensions may contain a thickening agent forexample beeswax, hard paraffin or cetyl alcohol. Sweetening agents, suchas those set forth above and flavoring agents may be added to provide apalatable oral preparation. These compositions may be perserved by theaddition of an antioxidant, such as ascorbic acid. Dispersible powdersand granules are also suitable for the oily suspension. The powders andgranules can be incorporated into the suspension with dispersing orwetting agents, suspending agents and one or more preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already mentioned above for the aqueous suspension.Additional excipients, for example sweetening agents or flavoringagents, may also be present.

[0070] The pharmaceutical composition can also be oil-in-wateremulsions. The oily phase of the emulsion can be an oil selected fromvegetable oil, for example, olive oil or arachis oil; a mineral oil, forexample, liquid paraffin; or a combination thereof. The emulsifyingagents can be naturally-occurring gums, phosphatides, esters or partialesters derived from fatty acids and hexitol anhydrides, and condensationproducts of said partial esters. Suitable emulsifying agents are gumacacia, gum tragacanth, soybean oil, lecithin, sorbitan monooleate,polyoxyethylene sorbitan monooleate. The emulsion can also containsweetening and flavoring agents including, in addition, syrups andelixirs such as glycerol, sorbitol, or sucrose, for example. Suchformulation can also contain soothing agents (demulcents),preservatives, flavoring agents, and coloring agents.

[0071] Aqueous and oily suspensions described for the oral dosage formscan be formulated into parenteral dosage, for example subcutaneously,intravenously, intramuscularly, intracisternally, or by infusion. Thesuspensions can be formulated using one or more non-toxic parenterallyacceptable diluents or excipients. Excipients suitable for the inventioninclude dispersing agents, wetting agents, suspending agents, and thelike. The suspension can be incorporated into a solvent vehicle, forexample 1,3-butane diol, water, Ringer's solution, isotonic sodiumchloride solution, and the like. In addition, sterile, fixed oils arealso suitably employed as a solvent or suspending medium. Examples ofoils for the parenteral composition are monoglycerides, diglycerides,and fatty acids, for example oleic acid.

[0072] Amyloid fibrils can be present anywhere in the body of themammal. Typically, amyloid fibrils aggregate in a particular organ, orpart of the organ, and form a film or mass of amyloid fibrils, calledamyloid plaque. Amyloid fibrils and amyloid plaque can be found, forexample, in the brain, pancreas, vasculature, spleen, liver, kidneys,adrenals, lymph nodes, muscle, cardiovascular system, skin, or anycombination thereof.

[0073] A significant advantage of the invention is the assessment of theamyloid fibrils localization and the ability to quantify the labeledamyloid fibrils. The localization or quantification of the labeledamyloid fibrils can be determined a variety of techniques. Examples areradioimaging, magnetic resonance imaging (MRI), single photon emissioncomputed tomographic imaging (SPECT), and other suitable detection orimaging techniques.

[0074] Overproduction of the readily aggregating amyloid protein cancause numerous disease including, but not limited to, Alzheimer'sdisease, Down syndrome, Type 2 diabetes mellitus, amyloid A (reactive),secondary amyloidosis, familial mediterranean fever, procalcitonin,Cruetzfeld-Jacob disease, bovine spongiform encephalitis, and the like.Moreover, mutations in the pathogenic genes believed to cause disease,for example, presenilin and APP genes, have been shown to cause anincrease in amyloid aggregation. Accordingly, the detection of amyloidaggregation, or the resulting amyloid plaque, is indicative ofamyloid-related pathogenic disease.

[0075] In accordance with the method of diagnosing a person having acondition associated with aggregated amyloid, the data is recorded andassessed by one with skill in the art of diagnosing or treatingamyloid-related disease. The method can be used for a wide variety ofamyloid-related disorders including, for example, but not limited to,Alzheimer's disease, Down syndrome, Type 2 diabetes mellitus, hereditarycerebral hemorrhage amyloidosis, amyloid A, secondary amyloidosis,familial Mediterranean fever, familial amyloid nephropathy withurticaria and deafness, amyloid lambda L-chain or amyloid kappa L-chain,A beta 2M, ATTR, familial amyloid cardiomyopathy, isolated cardiacamyloid, AIAPP or amylin insulinoa, atrial naturetic factor,procalcitonin, gelsolin, crytatin C, AApo-A-I, AApo-A-II,fibrinogen-associated amyloid; and Asor or Pr P-27 or in cases ofpersons who are homozygous for the apolipoprotein E4 allele, and thecondition associated with homozygosity for the apolipoprotein E4 allele.More particular examples of such diseases include, but are not limitedto, Dutch hereditary cerebral hemorrhage amyloidosis amyloid A,Muckle-wells syndrome, idiopathic-associated amyloid lambda L-chain,myeloma-associated amyloid lambda L-chain, macroglobulinemia-associatedamyloid lambda L-chain, idiopathic-associated amyloid kappa L-chain,myeloma-associated amyloid kappa L-chain, macroglobulinemia-associatedamyloid kappa L-chain, Portuguese familial amyloid polyneuropathy,Japanese familial amyloid polyneuropathy, Swedish familial amyloidpolyneuropathy, Danish familial amyloid cardiomyopathy, systemic senileamyloidosises, isolated atrial amyloid, medullary carcinoma of thethyroid, Finnish familial amyloidosis, Icelandic hereditary cerebralhemorrhage with amyloidosis, scrapie, Cruetzfeld-Jacob disease,Gertsmann-Straussler-Scheinker syndrome, and bovine spongiformencephalitis.

[0076] In some cases, the data obtained regarding the localization andquantification of the labeled amyloid fibrils can be compared to astandard, such as, for example, the localization and quantificationpatterns of a mammal having normal, or typical, levels of amyloid plaquerelative to like species. In accordance with a method of monitoring theaggregation of amyloid fibrils, the data obtained from the detection orimaging methods can be compared with earlier assessments of the amyloidcondition in the mammal as well as comparing the method to a standard todetermine the state of the amyloid aggregation or the progression of thedisease.

[0077] The data obtained also can be used in conjunction with a singledose or a regimen of therapy to determine the effect of the therapy onthe localization or quantification of the amyloid fibrils. For example,the radiolabeled compound can be administered to a patient undergoing acourse of amyloid-affecting therapy, such as doxorubicin, an organicmolecule. The radiolabeled compound interacts with the amyloid fibrilsto provide an amyloid-labeled complex, which can be used to detect,image, and preferably record, the condition of the affected organ atdesired periods in the therapy. The state of the amyloid aggregation andthe progress of the therapy can be suitably monitored.

[0078] In another example, the radiolabeled compound is administered toa mammal suspected of having an amyloid-related disorder. Theradiolabeled amyloid fibril complex is detected or imaged by thedescribed methods to obtain localization patterns and quantification ofthe labeled amyloid fibrils. The data obtained regarding the state ofthe amyloid aggregation can be recorded and compared with data obtainedfrom a normal, i.e. disease-free, brain to determine the state ofamyloid-affectedness in the patient. In this aspect, the radioactivecompound can be used as a biomarker for determining the relativecondition of a mammal potentially suffering from an amyloid-relateddisorder.

[0079] The preferred formulation for the compound is an oral dose form.More preferably, the compound is administered as a suspension, solution,or tincture. Dosage levels of the compound can be determined by one withskill in the art, considering the age, weight, and condition of thesubject to be treated, as well as the severity of the disease, thefrequency of the desired dosing, and the route of administration. Inparticular, the compound can be prepared in a single dose formulationfor the detection and imaging aspects of the invention. To provideguidance to the reader in carrying out the practice of the invention, anoral dose in the range of from about 10 to about 1000 milligrams perkilograms of body weight per day (mg/kg/day) are suitable for theinvention. The preferred dosage for the detection and imaging aspects ofthe invention are from about 250 to about 750 milligrams per kilogramsof body weight per day.

[0080] A method of the invention can also relate to treating a conditioncharacterized by aggregated amyloid fibrils. In this aspect, atherapeutically effective amount of a compound of formula (I) isadministered to the mammal, typically in a pharmaceutical carrier. Asufficient amount of time is allowed for the compound to interact withamyloid fibrils in the body. The interaction between the compound andthe amyloid fibrils inhibits amyloid protein fibrils from aggregatingand forming amyloid plaque. A “therapeutically effective” amount is theamount of active agent sufficient to inhibit or treat the condition.

[0081] The compounds also can be used to deliver treatment for anamyloid-related condition. The compound can be bound to a therapeutictreatment, typically a drug therapy, for example, synthetic ornaturally-derived organic compounds, proteins, antibodies, and the like.The compound, in combination with the therapeutic treatment, areadministered to a mammal, particularly a mammal in need of treatment foran amyloid-related condition. The compound interacts with the amyloidfibrils, transporting the target treatment to the site where thetreatment is needed. The compounds have demonstrated an affinity for theamyloid fibrils and, accordingly, deliver the desired treatmentefficiently and effectively to the target area, i.e. where the amyloidfibrils aggregate. Examples of active agents that can be monitoredinclude, but are not limited to, specific and non-specific drugtreatments such as proteins, peptides, carbohydrates, polysaccharides,glycoproteins, nucleic acids, antibodies, peptidomimetics, organicmolecules (preferably, less than 1500 kDa), fragments or recombinantforms of the above; inhibitors or activators of a molecule that isrequired for inhibiting, synthesizing, post-translation modification of,or functioning of some element involved in the localization orquantification of amyloid; regulators in the spatial or temporal controlof expression of a gene product; cytokines, growth factors, hormones,signaling components, kinases, phosphatases, homeobox proteins,transcription factors, translation factors, post-translational factorsand enzymes. Broad categories of drug therapies include, but are notlimited to, cholinesterase inhibitors, muscarinic agonists,anti-oxidants or anti-inflammatory agents. Examples of active agents foramyloid-related disorders are doxorubicin, galantamine, tacrine(COGNEX®), selegiline, physostigmine, revistigmin, donepizil (ARICEPT®),metrifonate, milameline, xanomeline, saeluzole, acetyl-L-carnitine,idebenone, ENA-713, memric, quetiapine, neurestrol and neuromidal.

[0082] In addition to methods for treating amyloidosis, the compoundsalso have demonstrated activity as an antioxidant. To enjoy theantioxidant properties of the invention, a quinolinehydrazone compound,for example a compound of formula (I), is administered to a patient,preferably in a pharmaceutically acceptable carrier as previouslydescribed. The antioxidant properties of the compound are especiallybeneficial for treating conditions characterized by oxidative damage inthe body, for example oxidative neuronal damage. Preferably, thecompound is administered as an oral or intravenous dosage form.

[0083] Dosing for the compound as a treatment agent, either for treatingamyloidosis or for treating a condition characterized by oxidativedamage, can be administered in any suitable manner. Examples of dosingregimens suitable for the invention are orally, parenterally,subcutaneously, intravenously, intramuscularly, intracisternally, or byinfusion, as described for the diagnostic aspect of the invention. Theprescribed dosing can be administered within the reasonable medicamentjudgment of the professional treating the patient, however, it issuggested that a suitable dose of the compound is in the range of fromabout 10 to about 1000 milligrams per kilograms of body weight per day.

[0084] The invention also relates to a method for detecting the presenceof aggregated prion protein in a mammal. The compound can beadministered orally as with the aspect of the invention related toamyloid-related detection and imaging. Prions, however, are more commonin animals. As such, it is preferred with the aspect of the inventionrelated to prion detection that a body fluid or tissue from the subjectmammal is obtained. The fluid or tissue is allowed to interact with thecompound to identify the presence of any aggregated prion. The presenceof the aggregated prion can be determined by the detection and imagingtechniques previously described. In this manner, the invention can bemore efficiently and effectively carried out for the practice ofdetecting and imaging the presence of aggregated prion in animals.Examples of prion-related disease, in particular, are generally diseasesthat affect livestock including, but not limited to, scrapie, a diseasecommon to sheep, and bovine spongiform encephalopathy (BSE), whichtypically affects cows and is more commonly known as mad cow disease.

[0085] As previously mentioned, human prion is less common than animalprion. Human prion has been reported in elderly patients and is believedto be transmitted to humans via BSE-infected beef. Accordingly, thequinolinehydrazone compounds can also be used to identify prionaggregation in humans.

[0086] Any body fluid extracted from the subject can be used for theassay. Typically, bodily fluid is meant to refer to fluids such as, forexample, lymph, blood, or urine. The preferred bodily fluid is lymph. Incertain preferred embodiments, the bodily fluid is filtered in vitrosuch that any prion infectious agent present in the bodily fluid doesnot pass through the filter. For example, a Millipore Ultrafree-MCpolysulfone membrane, 3000,0000 NMWL cutoff, can be used. The filter iscontacted with the compound. The presence of the resulting labeled prionprotein is determined using a SPECT detector, though any other methodknown to those skilled in the art to detect the labeled prion can alsobe used.

[0087] In another aspect, the invention relates to a complex comprisinga compound of formula (I) in association with or bound to the amyloidfibril or prion particle. As previously described amyloid fibril andprion particles have demonstrated properties indicative of or causingdisorders such as Alzheimer's and Crutzfeld-Jacob syndrome. By providinga compound of formula (I) and allowing the compound to associate with anamyloid fibril or prion, a complex of the compound with the amyloidfibril or prion is formed. The complex is useful for the identificationof the amyloid fibril or prion. For example, the complex can include aradiolabeled compound, which can be identified with a suitable detectionmethod.

[0088] The invention can be better understood in light of the followingexamples which are intended as an illustration of the practice of theinvention and are not meant to limit the scope of the invention in anyway.

EXAMPLE 1

[0089] Determination of In Vivo Brain Distribution: Intraveneous Dosing

[0090] Transgenic mice, designated “Tg”, were obtained. The transgenicmice carry the human gene for β-APP, a gene associated with β-amyloiddeposition in the brain. The mice were treated with thequinolinehydrazone compound,4-methyl-7-methoxy-2-(4-quinolylmethylhydrazino)quinoline. Dosing wasadministered either intravenously or orally. The intravenous dose wasadministered as a single injection (50 μmol/kg). Orally-treated animalsreceived a single dose (400 μmol/kg) via oral gavage, or were dosedtwice-a-day for 10 days (400 μmol/kg/dose/day).

[0091] Age-matched normal mice served as a negative control, i.e. noplaque formation in the animals was indicated. The control mice weretreated in the same manner as the transgenic mice, but with placebo.

[0092] In addition, a group of age-matched Tg mice were treated with thecarrier vehicle only. Age-matched Tg mice served as negative controlindicating that plaque was present, but no drug was administered.

[0093] All animals were sacrificed beginning 1, 3 and 10 h after thefinal dose. Fresh brains of the animals were removed, bisected, andpositioned in o-chlorotoluene (OCT). The brains were immersed into2-methylbutane, pre-cooled to 150° C. with liquid nitrogen. The brainswere cut into 20 μm thick sections at a temperature of −20° C. Brainsections were collected onto poly-L-lysine-coated glass microscopicslide and treated with (1 drop) of 75%, by volume (v/v), glycerin inpoly(butene-1-sulfone)(PBS). Each section was thawed briefly, coveredwith a coverslip, and examined immediately under a Zeiss LSM510 scanninglaser microscope (λ_(ex) is 488 nm; λ_(em) is greater than 520 nm) todetermine the extent and distribution of compound-associatedfluorescence.

[0094] The section adjacent to each compound-treated section of thebrain was stained with thioflavin S (Sigma Chemicals, Milwaukee, Wis.,U.S.A.). Sections stained with thioflavin S served as a positive controlfor identifying plaque. The distribution of compound-associatedfluorescence was compared with the distribution of thioflavin S-stainedsection in order to confirm whether the compound bound plaque.

[0095] All sections were examined 1 h later to assess artifactual drugdiffusion within thawed sections over time.

[0096] Adjacent brain sections of compound-treated mice displayedintense drug-related fluorescence (λ_(ex) is 365 nm; λ_(em) is 400 nm)in areas of plaque formation. The data confirm that thequinolinehydrazone compound is brain penetrable and preferentially bindsto human β-amyloid containing plaques.

[0097] All brain sections obtained from Tg mice receiving thequinolinehydrazone compound demonstrated marked plaque burden whencompared with sections stained by thioflavin S.

EXAMPLE 2

[0098] Dose-Dependent Pharmacokinetics in Plasma and BrainConcentrations

[0099] Dose-proportionality characteristics were assessed by measuringthe maxiumum concentration (C_(max)) for plasma and brain concentrationsafter oral dosing of normal mice. The mice were treated with six,individual, oral doses increased proportionally from 50 to 3,200μmole/kg. Plasma and brain concentrations were measured by HPLC at 0.5to 24 hours. The test results showed that C_(max) for plasma and brainincreases proportionally from 400 to 3,200 μmol/kg without evidence ofabsorption saturation.

EXAMPLE 3

[0100] In Vivo Determination of Plasma and Brain Exposure

[0101] Plasma and brain exposure were assessed by measuring area underthe curve (AUC) after oral dosing of normal mice. All mice were treatedwith six, individual, oral doses increased proportionally from 50 to3,200 μmole/kg. The data showed that plasma exposure increasedproportionally from 421 to 1,472 μmol/kg, but showed evidence ofsaturation at 3,024 μmol/kg. Brain exposure increased more rapidly inthe range between from 421 to 1,472 μmol/kg, but showed a similarsaturation phenomenon at higher doses.

[0102] The brain to plasma ratios were calculated and reported below.B/P Dose Plasma Brain Ratio μmole/kg AUC (0 to last) AUC (0 to last) AUC51 0.991 5.22 5.3 208 17 54.7 3.2 421 75.2 187 2.5 831 152 863 5.7 1472262 1490 5.7 3024 235 1714 7.3

[0103] The results summarized in the above table evidence goodpenetration of the compound to the brain tissue. As shown above, brainlevels average 5 times greater than plasma levels over the dose rangetested.

[0104] The terminal half-life of compound in the brain was measured todetermine the dosing frequency for 10-day tolerance studies. For thethree highest doses, brain terminal half-life ranged from 5-7 h.

EXAMPLE 4

[0105] Postmortem Staining of Amyloid Plaque

[0106] Brains from 12-14 month-old Tg mice were frozen and sectioned at−20° C. Each section was fixed in ice cold methanol (Aldrich ChemicalCo., Milwaukee, Wis.) for 10 minutes, rinsed in buffer and incubated in4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline for 90minutes at room temperature. Drug concentrations ranging from 0.01 M to10 M effectively demonstrated amyloid plaque in brains of Tg mice, butnot in brains of transgene-free mice. Fluorescence intensity in Tg mouseamyloid decreased with drug concentration. Staining of the adjacentbrains sections with thioflavin S confirmed that the compoundfluorescence does localize in amyloid plaques. The transgene mousebrains were confirmed to be plaque-free.

EXAMPLE 5

[0107] Determination of In Vitro Antioxidant Effect using NeuronalCytoprotection Model

[0108] The 4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinolinewas dissolved in 100 × stock of aqueous Locke's buffer to obtainconcentrations of 1, 3, 10, 30, and 100 μM solution. Neuronal cells wereincubated in each compound solution with and without ferrous ammoniumsulfate (200 μm) for 3 hours. High concentration supernatant wasobtained from each sample. The samples were analyzed under liquidchromatography/mass spectrometry (LC/MS). The quinoline demonstratedantioxidant properties.

EXAMPLE 6

[0109] Determination of In Vitro Antioxidant Effect using FerrousAmmonium Sulfate (FAS)-Induced Oxidative Toxicity Model

[0110] The 4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinolinewas tested in a primary neuronal model of FAS-induced lipid peroxidationtoxicity. The compound was treated in two manners. In one manner, thecompound was solubilized as 100× stock in 100% dimethyl sulfoxide (DMSO)to afford panel A. In another manner, the compound was solubilized inaqueous Locke's buffer to give panel B.

[0111] The data showed that the concentration of the drug dependentlyinhibited 200 μM FAS toxicity after 3 hours from 0.3-10 μM and showedclose to or complete neuronal protection at 10 μM when cell viabilitywas measured using ¹⁴C-AIB uptake or lactic dehydrogenase (LDH) release.Concentrations above 10 μM (30 and 100 μM) in 1% DMSO experimentsrevealed inherent drug toxicity and no neuronal protection against FASin panel A. In the is aqueous preparation of the compound there was muchless drug toxicity at 30 μM and considerable neuronal protection at 30and 100 μM, respectively. Brightfield photographs revealed the cellmorphology of the 10 μM+FAS cells to be no different than cells treatedwith no drug and no FAS (control cells).

[0112] The data suggested that antioxidant properties of the compoundcould enable the compound for having a dual pharmacology. Along with itsputative ability to inhibit plaque formation in Alzheimer's disease thecompound also could inhibit any oxidative neuronal damage associatedwith beta-amyloid.

[0113] The foregoing are intended to illustrate the invention and arenot meant to limit the scope of the invention, which is defined in theappended claims.

What is claimed is:
 1. A method for chemically tagging or inhibiting theaggregation of amyloid fibrils comprising the steps of: (a) providing acompound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and one or more atoms in the compound of formula (I) optionally isreplaced with a radiolabeled atom; and (b) allowing the compound toassociate with the amyloid fibrils.
 2. The method of claim 1 wherein theradiolabeled atom is selected from the group consisting of ³H, ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ¹⁸F, ¹⁹F, ¹⁵O, and ¹¹C.
 3. The method of claim 1wherein R¹, R², R³, R⁴, and R⁵ are each independently selected from thegroup consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl, n-hexyl, methoxy,ethoxy, isopropoxy, sec-butoxy, t-butoxy, phenyl, benzyl,trifluoromethyl, trifluoromethylether, and halo.
 4. The method of claim1 wherein the benzopyridinyl group for R⁶ is quinolyl or isoquinolyl. 5.The method of claim 1 wherein the compound of formula (I) in step (a) isincorporated in a pharmaceutically acceptable carrier.
 6. The method ofclaim 1 wherein the compound of formula (I) in step (a) is selected fromthe group consisting of:4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline; and4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.
 7. Themethod of claim 1 wherein the compound of formula (I) is4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinoline.
 8. Amethod for detecting an aggregation of amyloid fibrils comprising thesteps of: (a) providing a compound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and at least one atom in the compound is replaced with a radiolabeledatom; (b) allowing the compound to associate with the amyloid fibrils toprovide a labeled deposit; and (c) detecting the amount and location-ofthe labeled deposit.
 9. The method of claim 8 comprising the steps ofdetecting the labeled deposit by gamma imaging, magnetic resonanceimaging, or magnetic resonance spectroscopy.
 10. The method of claim 8further comprising the step of (d) evaluating or assessing the dataobtained in step (c) in an individual and optionally comparing the datawith analogous data obtained from a normal human or mammal to identify,assess, or diagnose the medical condition of the individual.
 11. Themethod of claim 10 comprising assessing the condition of an individualundergoing treatment for a condition characterized by the aggregation ofamyloid fibrils.
 12. The method of claim 11 wherein the condition isselected from the group consisting of Alzheimer's disease, Downsyndrome, Type 2 diabetes mellitus, hereditary cerebral hemorrhageamyloidosis, amyloid A, secondary amyloidosis, familial mediterraneanfever, familial amyloid nephropathy with urticaria and deafness, amyloidlambda L-chain or amyloid kappa L-chain, A beta 2M, ATTR, familialamyloid cardiomyopathy, isolated cardiac amyloid, AIAPP or amylininsulinoa, atrial naturetic factor, procalcitonin, gelsolin, crytatin C,AApo-A-I, AApo-A-II, fibrinogen-associated amyloid; and Asor or Pr P-27or in cases of persons who are homozygous for the apolipoprotein E4allele, and the condition associated with homozygosity for theapolipoprotein E4 allele; and the treatment comprises administering anactive agent selected from the group consisting of doxorubicin,galantamine, tacrine (COGNEX®), selegiline, physostigmine, revistigmin,donepizil (ARICEPT®), metrifonate, milameline, xanomeline, saeluzole,acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine, neurestroland neuromidal.
 13. The method of claim 8 wherein the compound offormula (I) is a biomarker for the aggregation of amyloid fibrils in anindividual.
 14. A method for treating a condition in an individualcharacterized by aggregation of amyloid fibrils comprising the steps of:(a) providing a compound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl,-aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; and said aryl groupsat each occurrence are optionally substituted with alkyl, alkoxy, orhalo; (b) allowing the compound to associate with the amyloid fibril;and (c) optionally repeating steps (a) and (b), as necessary, to improveor rehabilitate the condition of the individual.
 15. The method of claim14 wherein the compound of formula (I) is incorporated in apharmaceutically acceptable carrier.
 16. The method of claim 14 whereinthe condition is selected from the group consisting of Alzheimer'sdisease, Down syndrome, Type 2 diabetes mellitus, hereditary cerebralhemorrhage amyloidosis, amyloid A, secondary amyloidosis, familialMediterranean fever, familial amyloid nephropathy with urticaria anddeafness, amyloid lambda L-chain or amyloid kappa L-chain, A beta 2M,ATTR, familial amyloid cardiomyopathy, isolated cardiac amyloid, AIAPPor amylin insulinoa, atrial naturetic factor, procalcitonin, gelsolin,crytatin C, AApo-A-I, AApo-A-II, fibrinogen-associated amyloid; and Asoror Pr P-27 or in cases of persons who are homozygous for theapolipoprotein E4 allele, and the condition associated with homozygosityfor the apolipoprotein E4 allele.
 17. The method of claim 14 wherein thecondition is selected from the group consisting of Dutch hereditarycerebral hemorrhage amyloidosis amyloid A, Muckle-wells syndrome,idiopathic-associated amyloid lambda L-chain, myeloma-associated amyloidlambda L-chain, macroglobulinemia-associated amyloid lambda L-chain,idiopathic-associated amyloid kappa L-chain, myeloma-associated amyloidkappa L-chain, macroglobulinemia-associated amyloid kappa L-chain,Portuguese familial amyloid polyneuropathy, Japanese familial amyloidpolyneuropathy, Swedish familial amyloid polyneuropathy, Danish familialamyloid cardiomyopathy, systemic senile amyloidosises, isolated atrialamyloid, medullary carcinoma of the thyroid, Finnish familialamyloidosis, Icelandic hereditary cerebral hemorrhage with amyloidosis,scrapie, Cruetzfeld-jacob disease, Gertsmann-Straussler-Scheinkersyndrome, and bovine spongiform encephalitis.
 18. A method fordelivering a treatment to an individual for a condition characterized byan aggregation of amyloid fibrils comprising the steps of: (a) providinga composition comprising a compound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;in combination with an active agent; (b) administering the compositionto the individual; and (c) optionally repeating steps (a) and (b), asnecessary, to improve or rehabilitate the condition of the individual.19. The method of claim 18 wherein the active agent is selected from thegroup consisting of proteins, peptides, carbohydrates, polysaccharides,glycoproteins, nucleic acids, antibodies, peptidomimetics, organicmolecules, and fragments or recombinant forms thereof.
 20. The method ofclaim 18 wherein the active agent is selected from the group consistingof inhibitors or activators of a molecule that is required forinhibiting, synthesizing, post-translation modification of, orfunctioning of, some element involved in the localization orquantification of amyloid; regulators in the spatial or temporal controlof expression of a gene product; cytokines, growth factors, hormones,signaling components kinases, phosphatases, homeobox proteins,transcription factors, translation factors, post-translational factorsand enzymes, cholinesterase inhibitors, muscarinic agonists,anti-oxidants, and anti-inflammatory agents.
 21. The method of claim 18wherein the active agent is selected from the group consisting ofdoxorubicin, galantamine, tacrine (COGNEX®), selegiline, physostigmine,revistigmin, donepizil (ARICEPT®), metrifonate, milameline, xanomeline,saeluzole, acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine,neurestrol and neuromidal.
 22. A method for staining amyloid fibrilscomprising the steps of: (a) providing a compound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R ², R³, R⁴, and R⁵ are independently selectedfrom the group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group is of the formula —OR⁷, whereinR⁷ is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and one or more atoms in the compound of formula (I) is replaced with aradiolabeled atom; (b) applying the compound to a sample containingamyloid fibrils to form a labeled deposit; and (c) detecting the labeleddeposit.
 23. The method of claim 22 wherein the compound is incorporatedin a pharmaceutically acceptable carrier.
 24. A method for detectingamyloid deposits in biopsy or postmortem human or animal tissuecomprising the steps of: (a) incubating formalin-fixed biopsy orpostmortem human or animal tissue with a solution of a compound of theformula:

or a pharmaceutically acceptable salt, ester, solvate or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and one or more atoms in the compound of formula (I) is replaced with aradiolabeled atom; to provide a labeled deposit; and (b) detecting thelabeled deposit.
 25. A method for detecting the presence of aggregatedprion protein in a mammal, comprising the steps of: (a) extracting abodily fluid from the mammal; (b) contacting the bodily fluid with acompound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and one or more atoms in the compound of formula (I) is replaced with aradiolabeled atom; to provide a labeled deposit; and (c) detecting thelabeled deposit.
 26. A method for providing an antioxidant o anindividual, comprising administering a quinolinehydrazone compound tosaid individual.
 27. The method of claim 26, comprising administering acompound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; and said aryl groupsat each occurrence are optionally substituted with alkyl, alkoxy, orhalo.
 28. A complex comprising a compound of formula (I), or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof, inassociation with or bound to an amyloid fibril, wherein said compoundhas the formula:

wherein: R¹, R², R⁴, R⁴, and R⁵ are independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; and R⁶ is a benzopyridinyl group optionallysubstituted with one to three substituents selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; and said aryl groupsat each occurrence are optionally substituted with alkyl, alkoxy, orhalo.
 29. A complex comprising a compound of formula (I), or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof, inassociation with or bound to a prion, wherein said compound has theformula:

wherein: R¹, R², R³, R⁴, and R⁵ are independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; and R⁶ is a benzopyridinyl group optionallysubstituted with one to three substituents selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; and said aryl groupsat each occurrence are optionally substituted with alkyl, alkoxy, orhalo.
 30. A compound of the formula:

or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof, wherein: R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aryl,triflouromethyl, trifluoromethylether, halo, and a group of the formula—OR⁷, wherein R⁷ is alkyl or aryl; and R⁶ is a benzopyridinyl groupoptionally substituted with one to three substituents selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,trifluoromethylether, halo, and a group of the formula —OR⁷, wherein R⁷is alkyl or aryl; wherein said alkyl groups at each occurrence areoptionally substituted with alkoxy, aryl, or halo; said aryl groups ateach occurrence are optionally substituted with alkyl, alkoxy, or halo;and one or more atoms in the compound of formula (I) optionally isreplaced with a radiolabeled atom.
 31. The compound of claim 30 whereinR¹, R², R³, R⁴, and R⁵ are each independently selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, t-butyl, n-pentyl, t-pentyl, n-hexyl, methoxy, ethoxy,isopropoxy, sec-butoxy, t-butoxy, phenyl, benzyl, trifluoromethyl,trifluoromethylether, and halo.
 32. The compound of claim 30 wherein thebenzopyridinyl group for R⁶ is quinolyl or isoquinolyl.
 33. The compoundof claim 30 wherein the compound is selected from the group consistingof: 4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline; and4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.
 34. Thecompound of claim 30 wherein the radiolabeled atom is selected from thegroup consisting of ³H, ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ¹⁸F, ¹⁹F, ¹⁵O, and ¹¹C.